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Abstract:

The present invention provides a drug capable of initiating the
progression of the cell cycle of leukemia stem cells to overcome the
resistance of the leukemia stem cells to cell cycle-dependent
chemotherapeutic agents, and a drug for suppressing recurrence of
leukemia containing the same, and the like, an agent containing G-CSF,
wherein the agent is for inducing the progression of the cell cycle of
leukemia stem cells, a drug for suppressing recurrence of leukemia
containing a combination of G-CSF and a cell cycle-dependent antitumor
agent, and the like.

Claims:

1. (canceled)

2. The method according to claim 9, wherein the leukemia stem cells are
in the stationary phase.

3. The method according to claim 9, wherein the leukemia stem cells are
present in the niche in bone marrow.

4.-7. (canceled)

8. The method according to claim 12, which is for suppressing recurrence
of leukemia.

9. A method of inducing the progression of the cell cycle of leukemia
stem cells in a mammal, comprising administering G-CSF to the mammal.

10. A method of killing leukemia stem cells in a mammal, comprising
administering G-CSF and a cell cycle-dependent antitumor agent to the
mammal.

11. The method according to claim 10, wherein the cell cycle-dependent
antitumor agent is administered after administration of G-CSF.

12. A method of suppressing leukemia in a mammal, comprising
administering G-CSF and a cell cycle-dependent antitumor agent to the
mammal.

13. The method according to claim 12, wherein the cell cycle-dependent
antitumor agent is administered after administration of G-CSF.

14.-18. (canceled)

Description:

TECHNICAL FIELD

[0001] The present invention relates to a drug capable of initiating the
progression of the cell cycle of leukemia stem cells to overcome the
resistance of the leukemia stem cells to cell cycle-dependent
chemotherapeutic agents, and an agent for suppressing recurrence of
leukemia comprising the same, and the like.

[0003] Conventional chemotherapeutic agents have been posing the difficult
problem of being unable to rescue patients from AML because of its
recurrence after temporary remission. Therefore, to develop an effective
therapeutic agent and therapeutic method, there has been a strong demand
for elucidating the mechanism of recurrence by clarifying the properties
of leukemia, including the functional features and molecular features of
LSCs.

[0004] The present inventors have created a novel immunodeficient strain
with improved long-term xenogeneic engraftment,
NOD.Cg-PrkdcscidIl2rg.sup.tm1Wjl/J (NOD/SCID/IL2rgnull) mice,
carrying a complete null mutation (non-patent document 4) of the common
γ chain (non-patent document 5). This strain has life expectancy of
>90 weeks, and has been clarified to be able to more accurately assess
the engraftment and lymphoid/myeloid differentiation capacity of human
long-term repopulating HSCs (LT-HSCs) than strains such as NOD/SCID
(non-patent document 6), NOD/SCID/β2mnull (non-patent document
7), NOD-Rag1null (non-patent document 8) and
NOD-Rag1nullPrf1null (non-patent document 9) (non-patent
documents 10, 11).

[0005] The present inventors clarified that NOD/SCID/IL2rg KO mice
maintain leukemia engraftment rates higher than do NOD/SCID/b2m KO mice,
which are conventional immunodeficient mice becoming deficient not only
in the acquired immune system, but also in the innate immune system.
Furthermore, the present inventors showed that significantly higher
engraftment rates are maintained by transplanting the graft in the
neonatal stage than in the mature stage, which is used by many
researchers for its technical convenience. Also, the present inventors
found that recipient mice generated by transplanting LSCs derived from a
human acute myelogenous leukemia (AML) patient to neonatal
NOD/SCID/IL2rgnull mice well reproduced the pathologic condition of
AML in each human patient, and are appropriate as a mouse model of AML.
Furthermore, the present inventors found it possible to reproduce the
leukemic state observed in patient bone marrow and propagate human AML
cells (LSC and non-LSC), while maintaining the characters thereof, also
by performing secondary and tertiary transplantation of LSCs obtained
from a recipient mouse to another mouse. Furthermore, an analysis of the
mice revealed that LSCs home in an osteoblast-rich region (niche) of bone
marrow (BM) and engraft therein, where the LSCs have their cell cycle
ceasing in the stationary phase and are hence protected against apoptosis
induced by cell cycle-dependent chemotherapeutic agents (patent document
1, non-patent document 12). Therefore, it was thought that such LSCs
having their cell cycle stationary do cause leukemia recurrence after
chemotherapy.

[0006] By allowing cells in the stationary phase to initiate the
progression of the cell cycle thereof, and concurrently applying a cell
cycle-dependent chemotherapeutic agent, cell death such as due to
apoptosis can be induced. While some cases are known where cytokines were
allowed to act on a population of AML blast cells to reduce the
colonizing potential thereof in vitro (non-patent documents 13 to 16), no
investigation has been conducted to date to determine whether the effect
was LSC-specific. Nor has it been thought at all that the progression of
the cell cycle of LSCs as they are localized in the niche can be induced.

[0024] It is an object of the present invention to provide a method of
killing leukemia stem cells to suppress and prevent leukemia recurrence,
without relying on conventional chemotherapy alone, by initiating the
progression of the cell cycle of leukemia stem cells in the stationary
phase to make the leukemia stem cells sensitive to cell cycle-dependent
chemotherapeutic agents.

Means of Solving the Problems

[0025] As stated above, the present inventors elucidated that
chemotherapy-refractory leukemia stem cells are localized in the niche in
bone marrow (BM) (Nat Biotechnol 25, 1315-1321 (2007)), and that leukemia
stem cells have their cell cycle stationary in the niche. Hence,
mobilizing the cell cycle of leukemia stem cells in the niche is a key to
overcoming recurrence. With this in mind, the present inventors searched
for a drug capable of specifically initiating the progression of the cell
cycle of leukemia stem cells that have their cell cycle ceasing in the
stationary phase and cannot therefore be killed by cell cycle-dependent
chemotherapeutic agents, even in the niche, using the above-described
mouse model (NOD/SCID/IL2rgnull) of AML. As a result, the present
inventors discovered that by administering granulocyte colony stimulation
factor (G-CSF), initiation of the progression of the cell cycle of the
LSCs can be induced in the niche as well in vivo. Furthermore, the
present inventors demonstrated from a survival curve showing a
significant extension in transplantation experiments that by
administering in combination G-CSF and a cell cycle-dependent
chemotherapeutic agent, apoptosis of the leukemia stem cells localized in
the niche can be induced at extremely high efficiency, and, as a result,
leukemia recurrence can be prevented, and have completed the present
invention.

[0026] Accordingly, the present invention is as follows: [0027] [1] An
agent for inducing the progression of the cell cycle of leukemia stem
cells, which comprises G-CSF. [0028] [2] The agent according to [1],
wherein the leukemia stem cells are in the stationary phase. [0029] [3]
The agent according to [2], wherein the leukemia stem cells are present
in the niche in bone marrow. [0030] [4] A medicament for killing leukemia
stem cells, comprising a combination of G-CSF and a cell cycle-dependent
antitumor agent. [0031] [5] The medicament according to [4], wherein the
cell cycle-dependent antitumor agent is administered after administration
of G-CSF. [0032] [6] A drug for suppressing leukemia, comprising a
combination of G-CSF and a cell cycle-dependent antitumor agent. [0033]
[7] The drug according to [6], wherein the cell cycle-dependent antitumor
agent is administered after administration of G-CSF. [0034] [8] The drug
according to [6], which is for suppressing recurrence of leukemia. [0035]
[9] A method of inducing the progression of the cell cycle of leukemia
stem cells in a mammal, comprising administering G-CSF to the mammal.
[0036] [10] A method of killing leukemia stem cells in a mammal,
comprising administering G-CSF and a cell cycle-dependent antitumor agent
to the mammal. [0037] [11] The method according to [10], wherein the cell
cycle-dependent antitumor agent is administered after administration of
G-CSF. [0038] [12] A method of suppressing leukemia in a mammal,
comprising administering G-CSF and a cell cycle-dependent antitumor agent
to the mammal. [0039] [13] The method according to [12], wherein the cell
cycle-dependent antitumor agent is administered after administration of
G-CSF. [0040] [14] G-CSF for use in inducing the progression of the cell
cycle of leukemia stem cells. [0041] [15] A combination comprising G-CSF
and a cell cycle-dependent antitumor agent for use in killing leukemia
stem cells. [0042] [16] The combination according to [15], wherein the
cell cycle-dependent antitumor agent is administered after administration
of G-CSF. [0043] [17] A combination comprising G-CSF and a cell
cycle-dependent antitumor agent for use in suppressing leukemia. [0044]
[18] The combination according to [17], wherein the cell cycle-dependent
antitumor agent is administered after administration of G-CSF

Effect of the Invention

[0045] By using the agent for inducing the progression of cell cycle of
the present invention, it is possible to induce the progression of the
cell cycle of leukemia stem cells that are localized in the niche in bone
marrow (BM), and that have their cell cycle ceasing in the stationary
phase. Because leukemia stem cells having their cell cycle progressing
are more sensitive to cell cycle-dependent antitumor agents, it is
possible to kill leukemia stem cells at high efficiency by administering
in combination the agent for inducing the progression of cell cycle of
the present invention and a cell cycle-dependent antitumor agent. Because
leukemia stem cells are the major cause of leukemia recurrence, it is
possible to suppress and prevent leukemia recurrence by killing leukemia
stem cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0046] FIG. 1 is a graphic representation showing that administration of
G-CSF in vivo initiates the progression of the cell cycle of LSCs in the
stationary phase. (A) Representative contour maps generated by a flow
cytometric analysis of hCD34+CD38- LSCs of the BM at baseline
of recipients of primary transplantation of human AML in a constant state
without administration of a drug such as G-CSF, after administration of
cytarabine (Ara-C) in vivo, and after administration of G-CSF followed by
administration of cytarabine in vivo. (B) With administration of G-CSF in
vivo (open circles), the ratio of LSCs in the G0 phase of the cell cycle
in the recipient BM decreased compared with the absence of administration
of G-CSF (solid circles). Each horizontal bar indicates mean +SEM.
Two-tailed t-test revealed p<0.005 in each case.

[0047] FIG. 2 shows that initiation of the progression of the cell cycle
of AML cells that are present in the endosteal region is induced by
G-CSF. (A) Shown are representative examples of bone sections from
recipients of transplantation of human AML, derived from a recipient
receiving administration of G-CSF in vivo or recipient not receiving the
same, and immunohistochemically labeled with BrdU. This demonstrated that
in relation to administration of G-CSF, AML in the endosteal region
increases the uptake of BrdU (grey). (B) Immunofluorescence labeling with
Ki67, a marker of the progression of the cell cycle, demonstrated that
initiation of the progression of the cell cycle of AML cells in the
endosteal region is induced by administration of G-CSF. Shown are images
of CD34, Ki67, DAPI and a merged image thereof. Each scale bar indicates
20 μm (A) and 10 μm (B).

[0048]FIG. 3 shows that Ara-C-induced apoptosis is accentuated in the
endosteal region of BM by pre-administration of G-CSF. (A) Representative
histograms demonstrating that the expression of activated caspase-3 after
chemotherapy is accentuated by pre-administration of G-CSF in human
CD34+CD38- LSCs and CD34+CD38+ AML non-stem cells
derived from the BM of recipients of primary transplantation of human AML
after administration of Ara-C alone in vivo, and after administration of
G-CSF followed by administration of Ara-C in vivo. (B) In 7 recipients of
transplantation of each type of LSCs, the survival of LSCs decreased with
pre-administration of G-CSF followed by administration of Ara-C. Shown
are percentages of BM LSCs that were negative for activated caspase-3
(i.e., resistant to anticancer agents) when Ara-C was administered alone
(solid circles) or Ara-C was administered after administration of G-CSF
(open circles). Two-tailed t-test revealed a significant difference in
each case (p<0.05). (C) From HE staining and TUNEL staining of bone
sections from recipients of transplantation of AML, it is evident that
apoptosis is induced in the central region of BM with administration of
Ara-C alone, but cells adjoining to the endosteum survive (*). In
contrast, in the BM of recipients of administration of G-CSF followed by
administration of Ara-C, cell death due to apoptosis was shown in the
endosteal region (+), where treatment-refractory leukemia stem cells
engraft, as well as in the central region. Each scale bar indicates 10
μm.

[0049] FIG. 4 shows that by combining pre-administration of G-CSF and
administration of Ara-C, the frequency of LSCs is decreased and the
survival of secondary recipients is improved. (A) Since leukemia
recurrence/development has been proven to occur only from LSCs using the
maximum likelihood method, the frequency of LSCs was estimated by Poisson
statistics. In the analysis, positive transplantation was defined as
hCD45+>1.0% in peripheral blood on week 18 after transplantation.
*After administration, no sufficient number of hCD34+ cells for limited
dilution transplantation could be isolated. **Because engraftment
occurred in all recipients, frequency could not be estimated. ***Because
engraftment did not occur in any recipient, frequency could not be
estimated. P values were obtained by two-tailed t-test. The range
indicates +/- SEM. (B) The survival at large of mice receiving viable
hCD34+ AML cells derived from a recipient of transplantation of AML,
receiving administration of Ara-C alone or administration of Ara-C in
combination with G-CSF, was estimated by the Kaplan-Meier method.
Comparisons within each administration level and among different
administration levels, it was found that in secondary mouse recipients of
transplantation of AML receiving administration of Ara-C in combination
with G-CSF, the survival at large improved statistically significantly
(by log-rank test, p<0.0001). Dose 2×103 (solid line):
Ara-C alone n=25, G-CSF+Ara-C n=21; dose 2×104 (broken line):
Ara-C alone n=22, G-CSF+Ara-C n=14; dose 2×105 (broken line
with dots): Ara-C alone n=15, G-CSF+Ara-C n=14.

MODES FOR EMBODYING THE INVENTION

(1) Use of G-CSF for Inducing the Progression of the Cell Cycle of
Leukemia Stem Cells

[0050] The present invention provides an agent comprising G-CSF for
inducing the progression of the cell cycle of leukemia stem cells.

[0051] G-CSF is a publicly known cytokine, whose amino acid sequence and
the like are also publicly known. The G-CSF used in the present invention
is normally derived from a mammal.

[0052] Being "derived from a mammal" means that the amino acid sequence of
the G-CSF is a mammalian sequence. Mammals include, for example,
laboratory animals such as mice, rats, hamsters, guinea pigs, and other
rodents, and rabbits; domestic animals such as swines, cattle, goats,
horses, sheep, and minks; companion animals such as dogs and cats; and
primates such as humans, monkeys, cynomolgus monkeys, rhesus monkeys,
marmosets, orangutans, and chimpanzees. The G-CSF used in the present
invention is preferably derived from human.

[0053] Representative amino acid sequences of human G-CSF can include the
amino acid sequence shown by SEQ ID NO:2 (full-length) and SEQ ID NO:3
(mature type resulting from cleavage of signal sequence). Herein, for
proteins and peptides, the left end indicates the N-terminus (amino
terminus) and the right end indicates the C-terminus (carboxyl terminus),
according to the common practice of peptide designation.

[0054] Polypeptides that have a portion of the amino acid sequence of
natural type G-CSF deleted, substituted, added and/or inserted, and that
have granulocyte colony formation activity (G-CSF derivatives) are also
included in the G-CSF used in the present invention. Such G-CSF
derivatives are disclosed in, for example, Japanese Patent No. 2718426,
Japanese Patent No. 2527365, Japanese Patent No. 2660178, Japanese Patent
No. 2660179, JP-B-6-8317, Japanese Patent No. 2673099 and the like.

[0055] The G-CSF may be one isolated or purified from cells that produce
the same or a culture supernatant thereof by a protein separation and
purification technique known per se. The G-CSF may be a protein
biochemically synthesized using a chemical synthesis or cell-free
translation system, or may be a recombinant protein produced by a
transformant introduced with a nucleic acid having the base sequence that
encodes the aforementioned amino acid sequence.

[0056] It is preferable that the G-CSF used in the present invention have
been isolated or purified. "Isolated or purified" means that an operation
has been performed for removing components other than the desired
component. The purity of the isolated or purified G-CSF (G-CSF relative
to total polypeptide weight) is normally 50% by weight or more,
preferably 70% or more, more preferably 90% or more, most preferably 95%
or more (for example, substantially 100%).

[0057] The G-CSF used in the present invention may have been modified. The
modification is exemplified by, but is not limited to, addition of lipid
chain (aliphatic acylations (palmitoylation, myristoylation and the
like), prenylations (farnesylation, geranylgeranylation and the like) and
the like), phosphorylation (phosphorylation at serine residue, threonine
residue, tyrosine residue and the like), acetylation, addition of sugar
chain (N-glycosylation, O-glycosylation), addition of polyethylene glycol
chain, and the like.

[0058] A leukemia stem cell refers to a cell that meets the following
requirements: [0059] 1. Possesses the capability of causing leukemia in
living organisms selectively and exclusively. [0060] 2. Capable of
producing a leukemia non-stem cell fraction that cannot cause leukemia
per se. [0061] 3. Capable of engrafting in living organisms. [0062] 4.
Possesses a potential for self-replication.

[0063] Here, a potential for self-replication refers to the capability of
division such that one of the two cells resulting from cell division
becomes itself, i.e., a stem cell, and the other becomes a more
differentiated progenitor cell. The concept of leukemia stem cells is
already well established in the art and is widely accepted (D. Bonnet, J.
E. Dick, Nat. Med. 3, 730 (1997) T. Lapidot et al., Nature 367, 645
(1994)).

[0065] The leukemia stem cells to which the agent of the present invention
is applied are normally derived from a mammal. Mammals include, for
example, laboratory animals such as mice, rats, hamsters, guinea pigs,
and other rodents, and rabbits; domestic animals such as swines, cattle,
goats, horses, sheep, and minks; companion animals such as dogs and cats;
and primates such as humans, monkeys, cynomolgus monkeys, rhesus monkeys,
marmosets, orangutans, and chimpanzees. The leukemia stem cells used in
the present invention are preferably derived from a primate (for example,
humans) or rodent (for example, mice).

[0066] Human leukemia cells normally have the hCD45+hCD33+
phenotype. Of human leukemia cells, leukemia stem cells normally have the
hCD34+ phenotype. Of human leukemia stem cells, leukemia stem cells
that selectively have the capability of causing leukemia, that have their
cell cycle ceasing in the stationary phase, and that are resistant to
chemotherapeutic agents, normally have the hCD38- phenotype.

[0067] The cell cycle refers to the series of events that constitute cell
division, including mitosis, cytokinesis and interphases, in eukaryotic
organisms. In the cell, the first interphase (G1 phase) is followed by
the DNA synthesis phase (S phase), in which DNA synthesis takes place.
Upon completion of DNA synthesis, the second interphase (G2 phase) occurs
in preparation for cell division. After the preparation is ready and
genome replication is complete, the mitotic phase (M phase) occurs, in
which cell division begins. The cell proliferates to two cells having the
same genetic information, and returns to the first interphase (G1 phase).
If growth stimulation on the cells continue, the cells proceed to the DNA
synthesis phase (S phase), and the cell cycle is repeated. Without
stimulation, the cells remain in the stationary phase (G0 phase).

[0068] "Induction of the progression of the cell cycle" refers to allowing
cells in the stationary phase of the cell cycle to enter the cell cycle.
Therefore, by inducing the progression of the cell cycle, cell division
is initiated.

[0069] As shown in the Example below, the majority of leukemia stem cells
are present in the bone marrow niche (the endosteal surface adjoining to
a region where osteoblasts are abundantly present) and have their cell
cycle ceasing. Furthermore, stem cells entering the cell cycle are killed
by anticancer agents even if they have the phenotype
CD34+CD38-, which is characteristic of stem cells. Therefore,
it is critical in killing leukemia stem cells to cause the cells to leave
the stationary phase in the cell cycle thereof and enter the G1, S, G2, M
cycle. By applying G-CSF to leukemia stem cells, it is possible to allow
the leukemia stem cells to enter the cell cycle, or to raise the turnover
rate in the cell cycle, thereby to increase the sensitivity to cell
cycle-dependent antitumor agents. Therefore, the agent of the present
invention is useful as a medicament for increasing the sensitivity of
leukemia stem cells to cell cycle-dependent antitumor agents. As stated
below, by combining the agent of the present invention and a cell
cycle-dependent antitumor agent, it is possible to efficiently kill
leukemia stem cells.

[0070] The agent of the present invention can be administered as G-CSF as
it is, or in an appropriate pharmaceutical composition, to human or
non-human mammals (e.g., mice, rats, rabbits, sheep, swines, cattle,
cats, dogs, monkeys and the like). The pharmaceutical composition used
for the administration may comprise G-CSF and a pharmacologically
acceptable carrier, diluent or excipient. Such a pharmaceutical
composition is provided as a dosage form suitable for oral or parenteral
administration.

[0071] Examples of compositions for parenteral administration include
injections, suppositories and the like; the injection may include dosage
forms such as intravenous injections, subcutaneous injections,
intracutaneous injections, intramusclular injections, and drip
injections. Such an injection can be prepared according to a publicly
known method. Regarding how to prepare an injection, an injection can be
prepared, for example, by dissolving, suspending or emulsifying the
above-described G-CSF in a sterile aqueous liquid or oily liquid normally
used for injections. The aqueous liquid for injections is exemplified by
physiological saline, isotonic solutions containing glucose or other
auxiliary agent, and may be used in combination with an appropriate
solubilizer, for example, an alcohol (e.g., ethanol), a polyalcohol
(e.g., propylene glycol, polyethylene glycol), a nonionic surfactant
[e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of
hydrogenated castor oil)] and the like. The oily liquid is exemplified by
sesame oil, soybean oil and the like, and may be used in combination with
a solubilizer such as benzyl benzoate or benzyl alcohol. The injectable
preparation prepared is preferably filled in an appropriate ampoule. The
suppository to be used for rectal administration may be prepared by
mixing the above-described G-CSF in an ordinary suppository base.

[0072] Compositions for oral administration include solid or liquid dosage
forms, specifically tablets (including sugar-coated tablets and
film-coated tablets), pills, granules, powders, capsules (including soft
capsules), syrups, emulsions, suspensions and the like. Such a
composition is produced by a publicly known method, and may contain a
carrier, diluent or excipient in common use in the field of medicament
making. Useful carriers and excipients for tablets include, for example,
lactose, starch, sucrose, magnesium stearate and the like.

[0073] Also, the agent of the present invention may be formulated with,
for example, a buffering agent (for example, phosphate buffer solution,
sodium acetate buffer solution), a soothing agent (for example,
benzalkonium chloride, procaine hydrochloride and the like), a stabilizer
(for example, human serum albumin, polyethylene glycol and the like), a
preservative (for example, benzyl alcohol, phenol and the like), an
antioxidant and the like. The prepared medicament can be filled in an
appropriate ampoule.

[0074] The above-described pharmaceutical composition for parenteral or
oral administration is conveniently prepared in a medication unit dosage
form suitable for the dose of the active ingredient. Examples of such a
medication unit dosage form include tablets, pills, capsules, injections
(ampoules), aerosols and suppositories. Infusion pumps, transdermal
patches and subcutaneously embedded agents are also included as methods
of administration suitable for continuously obtaining a persistent drug
effect. Regarding the content of G-CSF, it is preferable that normally 1
to 5000 mg, particularly 2 to 3000 mg for injections, or 5 to 3000 mg for
other dosage forms, of the above-described G-CSF, per medication unit
dosage form be contained.

[0075] The dose of the above-described preparation containing G-CSF varies
depending on the recipient, symptoms, the route of administration and the
like; for example, when using the same to induce the progression of the
cell cycle of adult leukemia stem cells, it is convenient to administer
G-CSF normally at about 0.01 to 50 mg/kg body weight, preferably at about
0.1 to 20 mg/kg body weight, more preferably at about 0.2 to 10 mg/kg
body weight, based on a single dose, about 1 to 3 times a day, preferably
once a day, by intravenous injection or drip infusion. In the case of
other routes of parenteral administration (intramuscular administration,
subcutaneous administration) and oral administration, amounts according
to the above can be administered. In the case of a particularly severe
symptom, the dose may be increased according to the symptom. The dosing
frequency for G-CSF varies depending on the recipient, symptoms, the
route of administration and the like, and is, for example, a frequency of
once every 1 to 7 days, preferably a frequency of once every 1 to 3 days.
The number of times of administration of G-CSF varies depending on the
recipient, symptoms, the route of administration, the kind of antitumor
agent and the like, and is normally about 1 to 15 times, preferably 2 to
10 times.

(2) Combination of G-CSF and Cell Cycle-Dependent Antitumor Agent

[0076] The present invention further provides a medicament comprising a
combination of G-CSF and a cell cycle-dependent antitumor agent.

[0077] A cell cycle-dependent antitumor agent means an antitumor agent
that has a higher killing effect on cells having their cell cycle
progressing than on cells having their cell cycle ceasing, because the
active ingredient thereof targets a molecule or mechanism that is
contributory to the progression of the cell cycle. The cell
cycle-dependent antitumor agent is exemplified by, but is not limited to,
drugs that are publicly known as chemotherapeutic agents for cancer, for
example, alkylating agents (e.g., cyclophosphamide, iphosphamide and the
like), metabolism antagonists (e.g., cytarabine, 5-fluorouracil,
methotrexate and the like), anticancer antibiotics (e.g., Adriamycin and
the like, mitomycin), plant-derived anticancer agents (e.g., vinblastine,
vincristine, vindesine, taxol and the like), cisplatin, carboplatin,
etoposide and the like. In particular, cytarabine, 5-fluorouracil and the
like are preferred. Regarding "cell cycle-dependent antitumor agents",
detailed descriptions are given in, for example, a document, Brunton, L
L. Parker, K L. and Lazo, J S., Goodman and Gillman's The Pharmacological
Basis of Therapeutics. 11thed. McGraw Hill Publishing (2005), the
Wikipedia's entry "Anticancer Agents" and the like.

[0078] The cell cycle-dependent antitumor agent used in the present
invention is preferably one that is effective against leukemia
(particularly acute myelogenous leukemia).

[0079] When using G-CSF and a cell cycle-dependent antitumor agent in
combination, the dosing times of the G-CSF and cell cycle-dependent
antitumor agent are not limited; the G-CSF and cell cycle-dependent
antitumor agent may be administered to the recipient simultaneously or at
a time lag. The doses of the G-CSF and cell cycle-dependent antitumor
agent are not particularly limited, as far as the desired effect (killing
of leukemia stem cells or suppression and prevention of leukemia) can be
accomplished, and the doses can be chosen as appropriate according to the
recipient, the route of administration, symptoms, combination and the
like.

[0080] The mode of administration of G-CSF and a cell cycle-dependent
antitumor agent is not particularly limited, as far as the G-CSF and cell
cycle-dependent antitumor agent are combined at the time of
administration. Examples of such modes of administration include (1)
administration of a single preparation obtained by simultaneously
preparing G-CSF and a cell cycle-dependent antitumor agent, (2)
simultaneous administration via the same route of administration of two
different preparations obtained by separately preparing G-CSF and a cell
cycle-dependent antitumor agent, (3) administration at a time lag via the
same route of administration of two different preparations obtained by
separately preparing G-CSF and a cell cycle-dependent antitumor agent,
(4) simultaneous administration via different routes of administration of
two different preparations obtained by separately preparing G-CSF and a
cell cycle-dependent antitumor agent, (5) administration at a time lag
via different routes of administration of two different preparations
obtained by separately preparing G-CSF and a cell cycle-dependent
antitumor agent (for example, administration in the order of
G-CSF→cell cycle-dependent antitumor agent, or administration in
the reverse order) and the like.

[0081] The medicament of the present invention can be administered as a
combination of G-CSF and a cell cycle-dependent antitumor agent as they
are, or in an appropriate pharmaceutical composition, to human or
non-human mammals (e.g., mice, rats, rabbits, sheep, swines, cattle,
cats, dogs, monkeys and the like). The pharmaceutical composition used
for the administration may comprise G-CSF and/or a cell cycle-dependent
antitumor agent and a pharmacologically acceptable carrier, diluent or
excipient. Such a pharmaceutical composition is provided as a dosage form
suitable for oral or parenteral administration.

[0082] Examples of compositions for parenteral administration include
injections, suppositories and the like; the injections may include dosage
forms such as intravenous injections, subcutaneous injections,
intracutaneous injections, intramuscular injections and drip infusion
injections. Such an injection can be prepared according to a publicly
known method. Regarding how to prepare an injection, an injection can be
prepared by, for example, dissolving, suspending or emulsifying the
above-described G-CSF and/or cell cycle-dependent antitumor agent in a
sterile aqueous liquid or oily liquid normally used for injections. The
aqueous liquid for injections is exemplified by physiological saline,
isotonic solutions containing glucose or another auxiliary agent, and may
be used in combination with an appropriate solubilizer, for example, an
alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,
polyethylene glycol), a nonionic surfactant [(e.g., Polysorbate 80,
HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)] and
the like. The oily liquid is exemplified by sesame oil, soybean oil and
the like, and may be used in combination with a solubilizer such as
benzyl benzoate or benzyl alcohol. The injectable preparation prepared is
preferably filled in an appropriate ampoule. The suppository to be used
for rectal administration may be prepared by mixing the above-described
G-CSF and/or cell cycle-dependent antitumor agent in an ordinary
suppository base.

[0083] Compositions for oral administration include solid or liquid dosage
forms, specifically tablets (including sugar-coated tablets and
film-coated tablets), pills, granules, powders, capsules (including soft
capsules), syrups, emulsions, suspensions and the like. Such a
composition is produced by a publicly known method, and may contain a
carrier, diluent or excipient in common use in the field of medicament
making. Useful carriers and excipients for tablets include, for example,
lactose, starch, sucrose, magnesium stearate and the like.

[0084] Also, the medicament of the present invention may be formulated
with, for example, a buffering agent (for example, phosphate buffer
solution, sodium acetate buffer solution), a soothing agent (for example,
benzalkonium chloride, procaine hydrochloride and the like), a stabilizer
(for example, human serum albumin, polyethylene glycol and the like), a
preservative (for example, benzyl alcohol, phenol and the like), an
antioxidant and the like. The prepared medicament can be filled in an
appropriate ampoule.

[0085] The above-described pharmaceutical composition for parenteral or
oral administration is conveniently prepared in a medication unit dosage
form suitable for the dose of the active ingredient. Examples of such a
medication unit dosage form include tablets, pills, capsules, injections
(ampoules), aerosols and suppositories.

[0086] When G-CSF and a cell cycle-dependent antitumor agent are prepared
as separate preparations, the G-CSF content in the medicament of the
present invention is as described in the term (1).

[0087] The content of a cell cycle-dependent antitumor agent in the
medicament of the present invention differs depending on the form of the
preparation and the kind of antitumor agent, and is normally about 0.1 to
99.9% by weight, preferably about 1 to 99% by weight, more preferably
about 10 to 90% by weight, relative to the entire preparation.

[0088] When G-CSF and a cell cycle-dependent antitumor agent are used as a
single preparation prepared at the same time, the contents thereof may be
ones according to the above. In this case, the blending ratio of G-CSF
and the cell cycle-dependent antitumor agent can be chosen as appropriate
according to the recipient, the route of administration, symptoms, the
kind of cell cycle-dependent antitumor agent and the like.

[0089] The dose of G-CSF varies depending on the recipient, symptoms, the
route of administration and the like; for example, when G-CSF is used to
kill adult leukemia stem cells, it is convenient to administer G-CSF
normally at about 0.01 to 50 mg/kg body weight, preferably at about 0.1
to 20 mg/kg body weight, more preferably at about 0.2 to 10 mg/kg body
weight, based on a single dose, about 1 to 3 times a day, preferably once
a day, by intravenous injection or drip infusion. In the case of other
routes of parenteral administration and oral administration, amounts
according to the above can be administered. In the case of a particularly
severe symptom, the dose may be increased according to the symptom.

[0090] The dose of the cell cycle-dependent antitumor agent varies
depending on the recipient, symptoms, the route of m administration, the
kind of antitumor agent and the like; for example, when cytarabine is
used to kill adult leukemia stem cells, it is convenient to administer
cytarabine normally at about 0.01 to 2 g/kg body weight, preferably at
about 0.05 to 1 g/kg body weight, more preferably at about 0.1 to 0.5
g/kg body weight, based on a single dose, about 1 to 3 times a day,
preferably once a day, by intravenous injection or drip infusion. In the
case of other routes of parenteral administration and oral
administration, amounts according to the above can be administered. In
the case of a particularly severe symptom, the dose may be increased
according to the symptom.

[0091] The dosing frequency for G-CSF and/or the cell cycle-dependent
antitumor agent varies depending on the recipient, symptoms, the route of
administration, the kind of antitumor agent and the like, and is, for
example, a frequency of once every 1 to 7 days, preferably a frequency of
once every 1 to 3 days. The number of times of administration of G-CSF
and/or the cell cycle-dependent antitumor agent varies depending on the
recipient, symptoms, the route of administration, the kind of antitumor
agent and the like, and is normally about 1 to 15 times, preferably 2 to
10 times.

[0092] When the above-described G-CSF and cell cycle-dependent antitumor
agent are administered in combination as separately prepared
preparations, the preparation containing G-CSF and the preparation
containing the cell cycle-dependent antitumor agent may be administered
at the same time; however, the preparation containing the cell
cycle-dependent antitumor agent may be administered in advance, after
which the preparation containing G-CSF may be administered, or the
preparation containing G-CSF may be administered in advance, after which
the preparation containing the cell cycle-dependent antitumor agent may
be administered. When the same m are administered at a time lag, the time
lag differs depending on the active ingredient administered, dosage form,
and the method of administration; for example, when the preparation
containing G-CSF is administered in advance, a method is available
wherein the preparation containing the cell cycle-dependent antitumor
agent is administered within 1 minute to 3 days after administration of
the preparation containing G-CSF. When the preparation containing the
cell cycle-dependent antitumor agent is administered in advance, a method
is available wherein the preparation containing G-CSF is administered
within 1 minute to 3 days after administration of the cell
cycle-dependent antitumor agent.

[0093] Because leukemia stem cells are normally in the stationary phase
outside the cell cycle or have a slow turnover rate of the cell cycle, as
stated above, they exhibit resistance to cell cycle-dependent antitumor
agents. By applying G-CSF to leukemia stem cells, it is possible to allow
the leukemia stem cells to enter their cell cycle to thereby increase
their sensitivity to cell cycle-dependent antitumor agents. By allowing a
cell cycle-dependent antitumor agent to act on cells that have become
more sensitive to cell cycle-dependent antitumor agents, it is possible,
as a result, to kill leukemia stem cells at high efficiency. Therefore,
by administering the medicament of the present invention to a mammal
having leukemia stem cells, it is possible to kill the leukemia stem
cells in the mammal.

[0094] Based on this theory, it is preferable that administration of a
cell cycle-dependent antitumor agent take place simultaneously with
administration of G-CSF or after a given period following administration
of G-CSF, more preferably after a given period following administration
of G-CSF. Hence, the dosing protocol for the medicament of the present
invention preferably comprises a step for simultaneously administering
G-CSF and a cell cycle-dependent antitumor agent, or a step for
administering G-CSF and then administering a cell cycle-dependent
antitumor agent, more preferably comprises a step for administering G-CSF
and then administering a cell cycle-dependent antitumor agent. It is also
preferable that initiation of the progression of the cell cycle of
leukemia stem cells be confirmed after administration of G-CSF, and
thereafter a cell cycle-dependent antitumor agent be administered.

[0095] Therefore, the dosing protocol for the medicament of the present
invention preferably comprises the steps of: [0096] (1) administering
G-CSF and a cell cycle-dependent antitumor agent one time or a plurality
of times, [0097] (2) administering G-CSF one time or a plurality of times
in a first stage, and administering a cell cycle-dependent antitumor
agent one time or a plurality of times in a second stage, [0098] (3)
administering G-CSF one time or a plurality of times in a first stage,
and administering G-CSF and a cell cycle-dependent antitumor agent one
time or a plurality of times in a second stage, [0099] (4) repeating the
step (2) or (3) a plurality of times, and the like, [0100] more
preferably comprising any step selected from among (2) to (4) above.

[0101] In (2) and (3), the interval between the final administration in
the first stage and the final administration in the second stage varies
depending on the recipient, symptoms, the route of administration, the
kind of antitumor agent and the like, and is normally within 1 minute to
3 days.

[0102] More specific examples of the steps in the aforementioned dosing
protocol include, for example: [0103] (1) administering G-CSF and a cell
cycle-dependent antitumor agent at a frequency of once every 1 to 7 days,
preferably at a frequency of once every 1 to 3 days, 1 to 15 times,
preferably 2 to 10 times, [0104] (2) administering G-CSF at a frequency
of once every 1 to 7 days, preferably at a frequency of once every 1 to 3
days, 1 to 15 times, preferably 2 to 10 times, in a first stage, and
administering a cell cycle-dependent antitumor agent at a frequency of
once every 1 to 7 days, preferably at a frequency of once every 1 to 3
days, 1 to 15 times, preferably 2 to 10 times, in a second stage, [0105]
(3) administering G-CSF at a frequency of once every 1 to 7 days,
preferably at a frequency of once every 1 to 3 days, 1 to 15 times,
preferably 2 to 10 times, in a first stage, and administering G-CSF and a
cell cycle-dependent antitumor agent at a frequency of once every 1 to 7
days, preferably at a frequency of once every 1 to 3 days, 1 to 15 times,
preferably 2 to 10 times, in a second stage, [0106] (4) repeating the
step (2) or (3) a plurality of times, and the like.

[0107] Since leukemia stem cells are thought to a causal factor for
leukemia recurrence, it is possible to suppress and prevent leukemia
recurrence by using the medicament of the present invention. Hence, the
medicament of the present invention is useful as a drug for suppressing
leukemia (preferably a drug for suppressing recurrence of leukemia).
Recurrence of leukemia means that complete or partial remission of a
leukemia symptom by treatment is followed by re-growth of leukemia cells
resulting in re-emergence or aggravation of the leukemia symptom. It is
possible to suppress and prevent leukemia development (or recurrence) in
a mammal by administering the medicament of the present invention to the
mammal, wherein the mammal is at a risk of leukemia development (or
recurrence).

EXAMPLES

[0108] The present invention is hereinafter described in further detail by
means of the following Examples, by which, however, the invention is not
limited in any way.

(Materials and Methods)

Patient Samples

[0109] All experiments were performed with approval by the Institutional
Review Board for Human Research at RIKEN's RCAI. AML patient-derived
leukemia cells were collected with informed consent in writing. Samples
were derived from AML patients having the French-American-British (FAB)
classification system subtype M1 (not accompanied by maturation beyond
premyelocytic leukemia; case 4), M2 (myeloblastic, accompanied by
maturation; cases 3, 6, and 7), or M4 (myelomonocytic; cases 1 and 2).
BMMNCs (bone marrow mononucleate cells) were isolated using density
gradient centrifugation.

Mice

[0110] NOD.Cg-PrkdcscidIl2rg.sup.tmlWjl/Sz (NOD/SCID/IL2rgnull)
mice were developed at The Jackson Laboratory by backcrossing a complete
null mutation (Shultz, L. D. et al. Multiple defects in innate and
adaptive immunologic function in NOD/LtSz-scid mice. J Immunol 154,
180-191 (1995)) at the Il2rg locus onto the NOD.Cg-Prkdcscid
(NOD/SCID) strain. Mice were bred and maintained under defined flora with
irradiated food and acidified water at the animal facility of RIKEN and
at The Jackson Laboratory according to guidelines established by the
Institutional Animal Committees at the respective institutions.

Xenogeneic Transplantation

[0111] Newborn (within 2 days of birth) NOD/SCID/IL2rgnull recipient
received 150 cGy of total body irradiation using a 137Cs-source
irradiator, followed by intravenous injection of AML cells within two
hours. For primary transplantation, 103 to 5×104 sorted
BM cells per recipient from a 7AAD- lineage (hCD3/hCD4/hCD8)
-hCD34+hCD38- AML patient were used, as described in F.
Ishikawa et al., Nat. Biotechnol. 25, 1315 (2007). For secondary
transplantation after administration of Ara-C (cytarabine) alone or after
administration of G-CSF followed by administration of Ara-C,
2×102, 2×103, 2×104, or 2×105
sorted 7AAD-hCD45+hCD34+ BM cells per recipient were used.
For fluorescence-activated cell sorting, BMMNC cells from AML patients
were labeled with fluorescent dye-conjugated mouse anti-hCD3, anti-hCD4,
anti-hCD8, anti-hCD34 and anti-hCD38 monoclonal antibodies (BD
Immunocytometry), and BMMNC cells from recipients were labeled with mouse
anti-hCD45, anti-hCD34 and anti-hCD38 monoclonal antibodies (BD
Immunocytometry); the cells were sorted using FACSAria (Beckton
Dickinson, Calif.). Doublets were eliminated via analysis of
FSC/SSC-height and FSC/SSC-width. After the sorting, the purities of
hCD34+hCD38- cells and hCD34+ cells exceeded 98%.

Administration of G-CSF and Ara-C

[0112] For experiments involving administration of G-CSF alone,
administration of Ara-C alone, and administration of G-CSF followed by
administration of Ara-C, recipients of primary transplantation of human
AML were used 16 to 24 weeks after transplantation. For each experiment
for comparison of various dose groups, a pair of recipients was selected
from among litter mates, with the same primary AML sample transplanted in
the same amount on the same day so as to suppress variation among the
litter mates and variation due to differences in transplantation level.
Performed were administration of recombinant human G-CSF (Wako, Japan):
300 μg/kg s.c. qd×5 days; administration of Ara-C (Biogenesis,
Poole, UK): 1 g/kg i.p. qd×2 days; administration of G-CSF+Ara-C:
G-CSF 300 μg/kg s.c. qd×5 days, and concurrent administration of
Ara-C 1 g/kg i.p. qd×2 days on days 4 and 5 of administration. The
recipients were killed 16 hours after final injection. BrdU (1.5
mg/mouse; BD Biosciences, Calif.) was injected by i.p. to recipients
under a cell cycle analysis by BrdU uptake immediately after the final
injection (s.c. stands for subcutaneous administration, and i.p. for
intraperitoneal administration).

Flow Cytometry

[0113] For evaluation of human AML transplantation, blood was drawn from
the orbital sinus of each recipient every 3 weeks starting at week 6
after transplantation. Myelocytes were recovered from two tibiae and one
femur from each analyzed recipient; MNCs (mononucleocytes) were counted
manually and using an automated blood cell analyzer (Celltac α,
Nihon Kohden, Japan), and the absolute number of BMMNCs derived from each
recipient was estimated. The absolute number of human CD34+ cells
(derived from two tibiae and one femur) per mouse was determined by
multiplying the thus-obtained total BMMNC count by
7AAD-hCD45+hCD34+ BM cells (%). BrdU uptake was measured
using a BrdU flow kit (BD Pharmingen, Calif.). To quantify cells in the
G0 phase of the cell cycle, the cells were stained with Hoechst 33342 and
Pyronin Y, and then surface-stained using standard procedures.
Quantitation of cells undergoing apoptosis was performed by staining
activated caspase-3 in the cells using a rabbit anti-activated caspase-3
monoclonal antibody (BD Pharmingen, Calif.). Surface labeling was
achieved using mouse anti-human CD45, anti-CD34 and anti-CD38 monoclonal
antibodies (BD Immunocytometry). Analyses were performed using FACSAria
and FACSCanto II (Becton Dickinson, Calif.).

[0115] Differences in the ratios/absolute numbers of cells (%), activated
caspase-negative cells (%), and BM CD34+ cells in the cell cycle
were analyzed using two-tailed t-test (GraphPad Prism, GraphPad, San
Diego, Calif.). Differences in the number of viable cells were analyzed
by log-rank (Mantel-Cox) test (GraphPad Prism, GraphPad, San Diego,
Calif.). The frequency of LSCS was estimated by Poisson statistics using
the maximum likelihood method and two-tailed t-test with L-Calc software
(StemSoft Software, Vancouver, Canada).

Example 1

[0116] First analyzed was the status of the progression of the cell cycle
of LSCs and leukemia non-stem cells in the BM of NOD/SCID/IL2rgnull
recipients of transplantation of LSCs obtained from the BM of seven AML
patients. Although case-dependent variation existed, the ratios of cells
in the G0 phase and those in the G1 phase were significantly higher in
LSCs than in non-stem cells (hCD34+CD38+) in the BM of the
recipients (Table 1).

[0117] In the BMMNCs obtained from the recipients of AML transplantation,
CD34+CD38- LSCs and CD34+CD38+ AML non-stem cells
were compared. The results are shown as mean value +/- SEM; differences
were tested by two-tailed t-test.

[0118] Next, the relationship between the status of the progression of the
cell cycle of LSCs and the cytotoxic effect of the chemotherapeutic agent
cytarabine (Ara-C) was analyzed. When Ara-C was intraperitoneally
administered to NOD/SCID/IL2rgnull recipients of primary
transplantation of AML, CD34+CD38- AML cells in the S phase of
the cell cycle were selectively eliminated, whereas CD34+CD38AML cells in the G0/G1 phase were relatively highly resistant, and were
concentrated (% S=0.1+/-0.1 and % G0/G1=91.7+/-2.3 post-Ara-C, n=15;
two-tailed t-test compared with non-administration recipients revealed
p<0.0005; a representative data set of flow cytometry is shown in FIG.
1A).

[0119] Since CD34+CD38- AML cells having their cell cycle
progressing is selectively eliminated by Ara-C, it was hypothesized that
the sensitivities thereof to chemotherapeutic agents are increased by
inducing LSCs in the stationary phase to enter the cell cycle. To verify
this hypothesis, the effect of administration of granulocyte colony
stimulation factor (G-CSF) was analyzed in recipients of transplantation
of AML in vivo. While it is well described that the cell cycle is induced
by G-CSF in human and mouse HSCs, the effect of G-CSF on LSCs has not
been proven accurately. Therefore, first, an analysis was performed to
determine whether the status of the progression of the cell cycle of
CD34+CD38- LSCs changes with administration of G-CSF in
recipients of primary transplantation of AML in vivo. A representative
data set of flow cytometry is shown in FIG. 1A. In all cases examined, of
the LCSs of recipients receiving transplantation of AML given
administration of G-CSF, cells in the G0 phase fraction decreased
significantly, and concurrently LSCs in the S phase and G2/M phase
increased.

Example 2

[0120] The present inventors previously demonstrated that
CD34+CD38- LSCs are present selectively in the endosteal region
of BM, whereas CD38+ leukemia non-stem cells are detected mainly in
the central region of BM. It is important that LSCs adjoining to the BM
endosteum exhibit relatively high resistance to chemotherapy in vivo (F.
Ishikawa et al., Nat. Biotechnol. 25, 1315 (2007)). Therefore, to
directly evaluate the status of the progression of the cell cycle of LSCs
in the BM endosteal niche, histological analysis was performed on
recipients of primary transplantation of human AML (FIG. 2). In a
constant state without administration of a drug such as G-CSF, leukemia
cells in the central region of BM were strongly BrdU-positive; these
cells exhibited high proliferation capability, whereas AML cells
adjoining to the endosteum were found to be negative for BrdU staining;
it was shown that these cells did not have a vigorous progression of the
cell cycle (upper panel in FIG. 2A). In contrast, after administration of
G-CSF, as is seen by the increase in BrdU uptake, AML cells in the
endosteal region initiated the progression of their cell cycle (lower
panel in FIG. 2A). Likewise, immunofluorescence staining with Ki67, which
binds to a constituent of the nucleolus in the G1-S-G2 phase revealed
that in a constant state without administration of a drug such as G-CSF,
the majority of leukemia cells adjoining to the endosteum do not have a
vigorous progression of their cell cycle (upper panel in FIG. 2B).
Consistent with the finding of BrdU uptake assay in vivo, the expression
of Ki67 was induced in the AML cells in the BM center after 5 days of
administration of G-CSF, as well as in the AML cells in the endosteal
region (lower panel in FIG. 2B). These flow cytometric findings and
histological findings showed that G-CSF induces initiation of the
progression of the cell cycle in LSCs in the stationary phase that are
present in the endosteal niche.

Example 3

[0121] Next, to demonstrate that the sensitivity of LSCs to chemotherapy
increases with initiation of the progression of the cell cycle, an in
vivo model for evaluating the effects of administration of Ara-C alone
and administration of Ara-C following pre-administration of G-CSF on LSCs
in recipients of primary transplantation of AML was developed. After
administration of Ara-C alone or after administration of Ara-C following
pre-administration of G-CSF, the BM of the recipients was evaluated in
terms of 1) a flow cytometry fraction of activated caspase-3 positive
LSCs undergoing apoptosis, 2) histological localization of cells
undergoing apoptosis in the recipient BM as determined by TUNEL staining,
3) percentage and absolute number of remaining viable hCD34+ AML
cells, and 4) frequency and AML-causing potential of remaining LSCs in
alternative measurements of the likelihood of AML recurrence via limited
dilution and sequential transplantation of sorted hCD34+ cells. As
shown in FIG. 3A, with administration of Ara-C alone in vivo,
CD34+CD38+ AML non-stem cells underwent apoptosis, whereas the
majority of CD34+CD38- LSCs did not. In contrast, with
administration of G-CSF+Ara-C, the frequency of activated
caspase-3-negative LSCs decreased; it was shown that cell death due to
apoptosis increased (FIG. 3B). Although variation in this effect was
noted among the AML samples from the seven cases reflecting biological
heterogeneity among the cases (i.e., individual differences), a
statistically significant difference existed in that "leukemia stem cells
were unlikely to get killed when the anticancer agent was administered
alone, but a larger number of leukemia stem cells were killed by
mobilizing the cell cycle" in all cases. A concurrently performed direct
analysis of BM showed that with administration of Ara-C alone, the
recipients had TUNEL-negative AML cells remaining in the endosteum (FIG.
3C). However, with administration of G-CSF+Ara-C, as demonstrated by both
the reduction in cellularity revealed by HE staining and TUNEL staining
positivity in the remaining cells, more efficient cell death was observed
in the endosteum (and central region) in the recipients (FIG. 3C).

Example 4

[0122] To evaluate the frequency and function of LSCs remaining after each
dosing, limited dilution and secondary transplantation of living
hCD34+ BM cells, including leukemia stem cells sorted from
recipients given administration of Ara-C alone or G-CSF+Ara-C, were
performed. The absolute number of hCD34+ cells was obtained from the
number of mononucleocytes in two tibiae and one femur derived from each
recipient, and viable hCD34+ cell (%) was obtained by flow
cytometry. This demonstrated that in the BM of recipients given
administration of G-CSF+Ara-C, the number of viable hCD34+ cells
decreased significantly (Table 2).

[0123] The flow cytometric analysis of the BM obtained from the two tibiae
and one femur derived from recipients of transplantation demonstrated
that in the recipients of transplantation of AML with pre-administration
of G-CSF followed by administration of Ara-C, both the ratio and absolute
number of viable hCD45+CD34+ cells decreased. The results are
shown as mean value +/- SEM; differences were examined by two-tailed
t-test.

[0124] To definitely determine the function and frequency of LSCs
remaining after each administration, viable hCD34+ BM cells were
sorted, and re-transplanted to secondary recipients at doses of
2×102, 2×103, 2×104 and 2×105
cells per recipient (FIG. 4). The frequency of LSCs was estimated by
Poisson statistics, which is a standard methodology used to estimate the
frequency of HSCs by limited dilution (referring to a method wherein a
series of different numbers of stem cells are transplanted). As shown in
FIG. 4, the estimated frequency of LSCs that are causal cells for
recurrence was found to be significantly lower in the BM CD34+
population of the recipients given administration of G-CSF+Ara-C.
Furthermore, 24 weeks after transplantation, in the secondary recipients
of hCD34+ cells derived from a mouse receiving administration of
G-CSF+Ara-C, a statistically significant improvement in survival was
revealed at all doses (FIG. 4B). None of the secondary mouse recipients
with administration of Ara-C alone survived beyond 19 weeks after
transplantation, whereas 79.6% (39/49) of the secondary mouse recipients
receiving administration of G-CSF+Ara-C survived beyond 24 weeks after
transplantation; therefore, as leukemia stem cells were mostly killed,
and recurrence was significantly suppressed, by administration of
G-CSF+Ara-C, an efficacy of the present invention was demonstrated.

INDUSTRIAL APPLICABILITY

[0125] According to the present invention, it is possible to provide an
agent for suppressing recurrence of leukemia that dramatically improves
the therapeutic efficiency for leukemia, which is extremely intractable
so that the mean survival period, a patient prognostic factor, expected
with conventional standard therapies, is about 1 year.

[0126] This application is based on a patent application No. 2009-052723
filed Mar. 5, 2009 in Japan, the contents of which are incorporated in
full herein.